Abstract: Substance use disorder is a costly and debilitating chronic disorder, characterized by high rates of relapse despite successful attempts at rehabilitation and periods of abstinence. In addition, the rates of overdose and death associated with the opioid class of drugs have dramatically risen over the last decade and represent a major public health burden. To identify putative drug targets for the treatment of opioid use disorder, the molecular mechanisms that govern opioid seeking must be delineated. The goal of this proposal is to address this critical barrier in the field of substance abuse research by exploring entirely new mechanisms that modulate opioid seeking from a class of RNA called circular RNAs. Although circular RNAs have never been studied in drug seeking behaviors, this species of RNA has the potential to broadly influence cellular signaling at the epigenetic, transcriptional and translational levels, which in turn may impact a wide range of behavioral phenotypes. Circular RNAs are single-stranded transcriptional splice products that result from backsplicing of 3' to 5' ends of mRNA. Such splice products are beginning to be explored in the brain, with some expressed at higher levels than their mRNA counterparts, and have been reported to modulate critical processes including microRNA activity, gene expression, protein translation and even cognitive behaviors. We have identified a set of circular RNAs and their biogenesis enzymes that are regulated after heroin self-administration in the rat orbitofrontal cortex (OFC), a brain region essential to reward seeking. During the award period, we will explore the hypothesis that altered OFC circular RNA signaling contributes to opioid seeking phenotypes. We will use a multi-disciplinary approach to examine the cellular and behavioral consequences of heroin-induced circular RNA expression in a rat model of opioid seeking. First, heroin-associated circular RNAs will be genetically manipulated in the OFC to determine their contribution to motivation for opioids and relapse to opioid seeking after extinction or abstinence. We will then define the cellular phenotype of heroin-associated circular RNAs in the OFC for insights into the cellular circuits that have altered circRNA biogenesis as a result of heroin exposure. Finally, we will identify signaling pathways downstream of heroin-associated circular RNAs in the OFC to describe the molecular interactions of this novel class of RNA within drug-exposed neurons. Our dataset of heroin-associated circular RNAs have never been studied in the brain before and represent an entirely new area of research in the field of substance abuse. Therefore, the outcome of the unprecedented proposed studies will shed light on novel mechanisms that govern drug seeking and provide essential insight into the neurobiology of substance abuse.